Ring-truncated deguelin derivatives as potent Hypoxia Inducible Factor-1α (HIF-1α) inhibitors

Highlights

• A series of ring-truncated deguelin analogs were designed and synthesized as HIF-1α inhibitors.

• Compounds 25 and 33 exhibited potent HIF-1α inhibitions.

• Compound 25 showed significant antitumor and anti-angiogenic activities.

Abstract

A series of fluorophenyl and pyridine analogues of 1 and 2 were synthesized as ring-truncated deguelin surrogates and evaluated for their HIF-1α inhibition. Their structure–activity relationship was systematically investigated based on the variation of the linker B-region moiety. Among the inhibitors, compound 25 exhibited potent HIF-1α inhibition in a dose-dependent manner and significant antitumor activity in H1299 with less toxicity than deguelin. It also inhibited in vitro hypoxia-mediated angiogenic processes in HRMECs. The docking study indicates that 25 occupied the C-terminal ATP-binding pocket of HSP90 in a similar mode as 1, which implies that the anticancer and antiangiogenic activities of 25 are derived from HIF-1α destabilization by binding to the C-terminal ATP-binding site of hHSP90.

Introduction

Hypoxia Inducible Factor-1 (HIF-1) is the transcription factor that regulates the cellular response for the survival of cells in hypoxia. HIF-1 protein is a heterodimer that consists of a constitutively expressed β-subunit and an oxygen-regulated α-subunit. Under normoxia, HIF-1α is degradable by the pVHL-mediated ubiquitin protease pathway, which includes hydroxylation by prolyl hydroxylases (PHD), binding to the product of the von Hippel-Lindau (pVHL), being tagged with polyubiquitin and proteasomal degradation. However, under hypoxia, HIF-1α becomes stable from proline hydroxylation, accumulates and translocates to the nucleus. There, HIF-1α dimerizes with HIF-1β to activate the HIF-1 complex, which binds to hypoxia-response elements (HRE) in the HIF target genes to control transcription. This regulation can induce angiogenesis, proliferation, metastasis and invasion of cancer cells [1], [2]. Therefore, the HIF-1α inhibition can inhibit this angiogenesis, decrease the proliferation of cancer cells and reduce the chemotherapy resistance [3], [4], [5], [6], [7], [8], [9]. In addition, there has been growing interest in the biology of the HIF-1 pathway and its role in human diseases that are associated with the hypoxic micro-environment, such as cancer, stroke and heart disease [10], [11], [12], [13], [14], [15].

Heat shock protein 90 (HSP90) is a molecular chaperone that regulates the post-translational folding, stability and function of its client proteins such as ErbB2, Src, c-MET, AKT, Raf-1, MMP-2 and HIF-1α. Because the chaperone inhibition can induce the decomposition of HIF-1α, HSP90 inhibition is considered a new and effective therapy against angiogenesis-associated diseases such as cancer. Structurally, the HSP90 protein contains three functional domains: the ATP-binding, protein-binding, and dimerizing domain. Most HSP90 inhibitors that were developed as anticancer agents have been identified as the so-called N-term inhibitors, which bind to an ATP-binding domain in the N-terminal. However, these inhibitors cause some problems, and one of them induce a heat shock response (HSR), which ultimately leads to an increase in HSP90 and anti-apototic proteins such as HSP70 and HSP27. Therefore, the inhibition of another ATP-binding site in the C-terminal may be an alternative strategy to discover clinically applicable HSP90 inhibitors [16], [17], [18], [19], [20].

Deguelin, which is a naturally occurring rotenoid, has been reported to prevent tobacco carcinogen-induced lung carcinogenesis by blocking the Akt activation; it also exhibits potent apoptotic and antiangiogenic activities against diverse transformed cells and cancer cells in vitro (Fig. 1) [21](g), [21], [21](a), [21](b), [21](c), [21](d), [21](e), [21](f). It interferes with the chaperone function of HSP90 by inhibiting ATP binding, which induces the destabilization of HIF-1α and consequent tumor growth reduction in xenograft models of various human cancers [22].

Previously, Chang et al. reported that two ring-truncated deguelin analogues, compounds 1 and 2, exhibited excellent HIF-1α suppression and potent cell growth inhibition in the human non-small-cell lung carcinoma cell line, H1299 (Fig. 1) [23]. In addition, their in vivo antiangiogenic activities were observed in the zebrafish model in a dose-dependent manner. Jo et al. also reported that the destabilization of HIF-1α by both compounds suppressed hypoxia-mediated retinal neovascularization and vascular leakage in diabetic retina without inducing a definite toxicity in the oxygen-induced retinopathy mouse model [24]. The results indicated that the new HSP90 inhibitors 1 and 2 were considered promising lead compounds for anti-proliferation and anti-angiogenesis.

Structurally, the ring-truncated deguelin scaffold was divided into three pharmacophoric parts: A-region (3,4-dimethoxyphenyl), B-region (linker), and C-region (2,2-dimethyl chromene ring) (Fig. 2). To further optimize the leads 1 and 2 as anticancer agents, we decided to investigate their fluorophenyl (X = C–F) and pyridine (X = N) derivatives in the A-region to improve the target binding for the HIF-1α inhibition and aqueous solubility. We presumed that polarizing the 3,4-dimethoxyphenyl group by incorporating a polar nitrogen or fluoro atom might provide better binding interaction to HSP90 and pharmacokinetic profile. In addition, α-methyl carbonyl in the B-region was modified with its bioisosteres, which include olefin, diol, alcohol, and acyl groups (Fig. 2).

In this paper, we investigated the structure activity relationships of the fluorophenyl and pyridine analogues of 1 and 2 for HIF-1α inhibition using western blot assay. With selected potent inhibitors in the series, we further characterized their cytotoxicities in a tumor cell line and anti-angiogenesis in hypoxia-mediated angiogenic processes in human retinal microvascular endothelial cells. In addition, we performed the docking study with HSP90 to determine its mode of action.